The core of the cell death machine are members of a family of proteases known as caspases. Caspases become activated in response to many different death signals. They then cleave a number of different cellular substrates, leading ultimately to cell death. Most if not all cells constitutively express caspase zymogens (inactive precursors) sufficient to bring about apoptosis. Thus, the key to cell death and survival signaling revolves around controlling the levels of active caspases in the cell. Deregulation of caspase activation, and thus cell death, is associated with many human diseases. For example, inappropriate inhibition of caspase activity is associated with cancer and autoimmunity, while ectopic caspase activation is associated with many neurodegenerative diseases. The only known cellular caspase inhibitors are members of the Inhibitor of Apoptosis (lAP) family of proteins, which therefore constitute a last line of defense against caspase activation. Because of their critical role, determining how lAPs function to inhibit cell death, and how these activities are regulated, is important for understanding how cell death is regulated in health and disease. In this proposal we describe genetic and biochemical experiments designed to identify pathways by which lAP-dependent inhibition of cell death is regulated. Our efforts are focused on several goals - the identification of upstream activators of caspase activity that the Drosophila lAP DIAP1 must fight against to guarantee cell survival, and the identification and characterization of proteins that directly or indirectly alter the ability of DIAP1 to inhibit apoptosis. A major focus of these latter efforts will be to identify the mechanisms by which cell death activators tip the cellular balance towards death by promoting lAP degradation. These efforts will involve a characterization of the roles of known regulators of lAP stability. In addition, we will use affinity purification of lAP-binding proteins from cell culture, as well as a newly developed genetic screen to identify unknown regulators of lAP function. Finally, we will also characterize the roles and mechanism of action of a second Drosophila lAP, DIAP2.